MX2011000306A - Salts of hiv inhibitor compounds. - Google Patents

Abstract

The invention is related to salts of anti-viral compounds, compositions containing such salts, and therapeutic methods that include the administration of such salts, as well as to processes and intermediates useful for preparing such salts.

Description

SALTS OF HIV INHIBITORS COMPOUNDSField of the InventionThe present invention relates generally to salts of compounds with antiviral activity and more specifically with HIV properties.

Background of the InventionHuman immunodeficiency virus (HIV) is a retrovirus that can lead to acquired immune deficiency syndrome (AIDS), a condition in humans, in which the immune system is weakened, leading to life-threatening opportunistic infections. HIV inhibitors are useful for treating an HIV infection in a mammal (e.g., reducing and limiting the establishment and progress of HIV infection) as well as in diagnostic assays for HIV. The utility of currently commercialized HIV inhibitors is to some extent limited by toxicity and other side effects. Therefore, there is a need for new HIV therapeutic agents.

A pharmaceutical formulation of a therapeutic agent, must provide reproducibly and consistently the therapeutic agent to a patient in need thereof. This supply consistency can be achieved, at least in part, by the incorporation of a stable, soluble, solid state form of the therapeutic agent into the pharmaceutical composition. In addition, the synthesis of the desired solid state form of the therapeutic agent should be technically and economically feasible, and should be suitable for large-scale commercial production.

Brief Description of the InventionThe N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate, is a reverse transcriptase inhibitor that blocks replication of HIV viruses, in vivo, and in vitro, and has limited undesirable side effects when administered to humans. The structure of N - [(S). { . { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl is shown in the formula P:The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy .) methyl) phenoxyphosphinoyl] -L-alaninate is an amorphous, low-melting solid that is difficult to isolate, purify, store for a prolonged period and formulate as a pharmaceutical composition. The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate is also a weak base having the ability to form salts with acids. Accordingly, in one aspect, the present invention provides stable salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro Ethyl -2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphino] -L-alaninate, which are more physically stable, and are more readily isolated and formulated, than the free base form of the compound.

The salts of the present invention are useful, for example, to treat human patients infected with human immunodeficiency virus (strains of HIV-1 or HIV-2) that cause AIDS. Salts of the present invention are also useful, for example, for preparing a medicament for treating HIV or a disorder associated with HIV. Salts of the present invention are also useful, for example, to inhibit replication of HIV viruses in vitro, and can therefore be used in biological assays as a control compound to identify other reverse transcriptase inhibitors, or investigate the mechanism of action of HIV reverse transcriptase and its inhibition.

Therefore, in one aspect, the present invention provides citrate, succinate and malonate salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9- il) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate, and methods for making the above salts. In some embodiments, the salts of the present invention are anhydrous,although in other embodiments the salts of the present invention are at least partially hydrated. In some embodiments, the salts of the present invention exist as crystalline forms.

Therefore, the present invention comprises citrate, succinate and malonate salts of the compound of formula P, as well as hydrates thereof. The hydrates of the present invention may be in a partially hydrated (eg, hemi-hydrate) or total (eg, mono-hydrate) state. The present invention also comprises the salts in question in anhydrous and essentially anhydrous states. Similarly, the salts of the present invention and the hydrates thereof comprise amorphous and crystalline states, as well as states comprising both amorphous and crystalline characteristics. As used in the present invention, the term "crystalline" means a material having a long-range, ordered molecular structure. In contrast, "amorphous" materials do not have a long range order. It will be understood that crystalline materials are generally more thermodynamically stable than amorphous forms of the same substance. Therefore, with only some notable exceptions, it is generally preferred to use crystalline materials in pharmaceutical applications. A measure of the degree of crystallinity of the compounds of the present invention can be seen, for example, in the sharpening of the absorption bands DSC and XRPD (peaks). The sharper the peak, the greater the degree of crystallinity. Conversely, the wider the peak, the lower the degree of crystallinity.

As described in greater detail in Example 12 in the present invention, in specific embodiments, a citrate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H -purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphino] -L-alaninate is characterized by absorption bands, obtained from a diffraction pattern of X-ray powder, at spectral d-spacings of 4.48, 3.12 and 6.05 angstroms; a N- [(S) ( { [(2R, 5R) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] oxy] methyl) phenoxyphosphinoyl] -L-alaninate succinate salt is characterized by absorption bands obtained from a spectral X-ray powder diffraction pattern of 3.57, 4.80 and 4.99 angstroms; and a malonate salt of N - [(S). { [(2R, 5R) -5-. { 6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl is characterized by absorption bands, obtained from an X-ray powder diffraction pattern, at spectral-d-spacings of 4.99, 5.93 and 4.72 angstroms. In another specific embodiment, the present invention provides a citrate salt of N - [(S) ( { [(2R.5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro Ethyl -2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate having a melting point of 142 ° C to 150 ° C.

The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate is a prodrug of amidate which undergoes reaction with decomposition in protic solvents. The reaction range depends on the pH and temperature. Consistently, the formation of a stable salt between the amidate prodrug and the citric acid, succinic acid and / or malonic acid, which each contain nucleophilic portions with the ability to react with the prodrug (for example, reaction with the of prodrug amidate) is a surprising and unexpected result.

In another aspect, the present invention provides pharmaceutical compositions that each include a therapeutically effective amount of a salt of the present invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions of the present invention can also include an additional therapeutic agent, such as an antiviral, antibacterial, antifungal or anti-cancer agent. The pharmaceutical compositions of the present invention may be in the form of unit dosage forms, such as tablets or capsules. Unit dosage forms usually provide an effective daily dose of a salt of the present invention to a human being in need thereof. The effective daily doses of the salts of the present invention normally range from 1 mg to 100 mg, such as1 O mg to 30 mg.

In another aspect, the present invention provides methods for making the citrate, succinate and malonate salts of the present invention. Thus, for example, the present invention provides a process for preparing a citrate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9- M) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate, wherein the process includes the step of contacting about one equivalent of the free base of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy}. methyl) phenoxyphosphinoyl] -L-alaninate in a suitable solvent (eg, acetonitrile) with from about one equivalent to about 1.2 equivalents of citric acid at a temperature in the range of about 55 ° C to about 75 ° C.

The present invention also provides a process for preparing a succinate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro -2,5-dihydrofuran-2-yl) oxy} methyl) phenoxyphosphinoyl] -L-alaninate, wherein the process includes the step of contacting about one equivalent of a free base of N - [(S). { . { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate in a suitable solvent (eg, 2-butanone) with from about one equivalent to about 1.2 equivalents ofsuccinic acid at a temperature in the range of about 60 ° C to about 70 ° C to form the succinate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H -purin-9-yl) r4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate. In addition, the present invention provides a process for preparing a malonate salt of N - [(S). { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl, wherein the process includes the step of contacting about one equivalent of free base of N - [(S) ( { [(2R, 5R) -5- (6-amino -9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate in a suitable solvent (for example, 2-butanone) with from about one equivalent to about 1.2 equivalents of malonic acid at a temperature in the range of about 50 ° C to about 70 ° C to form the malonate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate ethyl.

In a further aspect, the present invention provides methods for treating or preventing prophylactically AFDS, wherein the methods include the step of administering to a human being suffering from AIDS a therapeutically effective amount of a salt of the present invention, or a hydrate thereof.

Brief Description of the FiguresFigure 1 shows a characteristic differential scanning calorimetry (DSC) trace for the malonate salt of N - [(S). { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxi} methyl) phenoxyphosphinoyl] -L-alaninate ethyl.

Figure 2 shows a characteristic DSC trace for the succinate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4- fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) -phenoxyphosphinoyl] -L-alaninate.

Figure 3 shows a characteristic DSC trace for the citrate salt of N - [(S) ([[(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro- Ethyl 2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

Figure 4 shows a characteristic X-ray powder diffraction pattern (XRPD) characteristic for the malonate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H- purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate.

Figure 5 shows a characteristic XRPD pattern for the succinate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-M) - 4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

Figure 6 shows a characteristic XRPD pattern for the citrate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4- fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

Detailed description of the inventionWhen commercial names are used in the present invention, the applicants intend to independently include the product under their trade name, and the active pharmaceutical ingredient (s) of the product of the commercial name.

N-α salts (SUf r (2R.5R)) - 5- (6-amino-9H-purin-9-in-4-fluoro-2,5-dihydrofuran-2-yloxy.) Metillfenoxifosfinoill- Ethyl L-alaninateIn one aspect, the present invention provides N- [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl.) Salts of citrate, succinate and malonate. Ethyl 4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

The citrate salt is shown in formula I:The succinate salt is shown in formula II:IIThe malonate salt is shown in the formulaneither ,Described in Example 1 in the present invention, a method for synthesizing N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4- fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate. The methods for making the malonate, succinate and citrate salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro- 2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate are described in Examples 3, 4 and 5, respectively. Some physical properties of the above salts are described in Example 6 of the present invention, and demonstrate, for example, that each of these salts is physically stable when stored at a temperature of 40 ° C and a relative humidity of 75% .

The citrate, succinate and malonate salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5 ethyl -dlhydrofuran-2-ll] oxy} methyl) phenoxyphosphinoyl] -L-alaninate are useful, for example, to inhibit replication of HIV in vitro and in vivo. In this regard, as explained in more detail in Example 8 of the present invention, the N - [(S). { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate is a prodrug that is metabolized in the human body to produce a parent compound, which in turn is phosphorylated within the body to produce an active metabolite that inhibits replication of HIV. Example 8 of the present invention shows that N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5 -dihydrofuran-2-yl] oxy.} methyl) phenoxyphosphinoyl] -L-alaninate causes greater accumulation of the active metabolite in the white blood cells, which are the cells harboring the HIV virus, than the compound of origin . In addition, Example 9 of the present invention presents in vitro data showing that N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4 -fluoro-2,5-dihydrofuran-2-yl] oxy.} methyl.} phenoxyphosphinoyl] -L-alaninate ethyl is an anti-HIV drug more potent than the parent compound as evaluated in an in vitro assay. In addition, Example 10 of the present invention provides data showing that a tablet containing the citrate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H -purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-il] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl provides this drug to the bloodstream of Beagle dogs with pharmacokinetics similar to the liquid preparation of the drug administered orally. Therefore, the citrate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5- dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate is a physically and chemically stable material composition that can be administered orally to a living subject, to provide a therapeutically effective amount of N- [ (S) { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl, which is an anti-HIV agent more effective than the compound of origin.

Pharmaceutical CompositionsIn another aspect, the present invention provides pharmaceutical compositions, also referred to as pharmaceutical formulations that include a therapeutically effective amount of one or more salts of the present invention, and a pharmaceutically acceptable carrier or excipient.

Although it is possible for the salts of the present invention to be administered alone, it is usually preferable to administer it as pharmaceutical compositions. The pharmaceutical compositions of the present invention are formulated with conventional carriers and excipients, which areThey select according to ordinary practice. The transporter (s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically safe for the recipient thereof. The tablets contain components such as excipients, glidants, fillers, linkers and the like. The aqueous formulations are prepared in sterile form, and when projected for delivery through a different administration to oral administration, they are generally isotonic. All formulations optionally contain excipients such as those set forth in the Pharmaceutical Excipients Manual (R.C. Rowe et al., Pharmaceutical Press, 5th edition, 2006). The excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is usually from about 7 to 10.

The formulations can be conveniently presented in a unit dosage form (e.g., tablets) and can be prepared by any of the methods known in the pharmaceutical art. The techniques and formulations are generally found in Reminaton's Pharmaceutical Sciences Publication (Mack Publishing Co., Easton, PA). Said methods include the step of associating the active ingredient with the carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and thoroughly associating the active ingredient with liquid carriers or finely divided solid carriers or both, and subsequently, if necessary, shaping the product.

The pharmaceutical formulations containing the active ingredient may be in any form suitable for the method of projected administration. When used for example for oral use, tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs can be prepared. The compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and said compositions can contain one or more agents including sweetening agents, flavoring agents, coloring agents and preservatives, with the object to provide a good tasting preparation. Tablets containing active ingredient in mixtures with pharmaceutically acceptable non-toxic excipients which are suitable for tablet manufacture are also acceptable. These excipients may be, for example, inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulation and disintegration agents such as, corn starch, or alginic acid; binding agents such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may not be coated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may also be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oily medium such as coconut oil, liquid paraffin or olive oil.

The aqueous suspensions of salts of the present invention contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include a suspending agent such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum, and dispersing or wetting agents such as naturally occurring phosphatide, (e.g., lecithin. ), a condensation product of an alkylene oxide with a fatty acid (for example, polyoxyethylene stearate), a condensation product of ethylene oxide, a long-chain aliphatic alcohol (for example, heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ether derived from a fatty acid and a hexitol anhydride (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl benzoate or p-hydroxy n-propyl, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oral suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.

These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

The dispersible powders and granules of the present invention suitable for the preparation of an aqueous suspension by the addition of water, provide the active ingredient in mixtures with a wetting or dispersing agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified with those described above. Additional excipients, for example, sweetening, flavoring and coloring agents may also be present.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as acacia gum, and tragacanth gum, naturally occurring phosphatides such as soybean lecithin, esters, partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate , and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents such as glycerol, sorbitol or sucrose. Said formulations may also contain an emollient, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the present invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known technique using the appropriate dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butane diol or prepared as a lyophilized powder. Among the vehicles and acceptable solvents that can be used are water, Ringer's solution and an isotonic sodium chloride solution. In addition, the sterile fixed oils can be conventionally mixed as a solvent or suspension medium. For this purpose, any soft fixed oil including synthetic mono or diglycerides can be employed. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables.

The amount of active ingredient that can be combined with the carrier material to produce a simple dosage form will vary depending, for example, on the particular mode of administration. For example, a time release formulation projected for oral administration to humans, may contain from about 3 to 1000 mg of the active material in compound with a suitable and convenient amount of carrier material, which may vary from about 5 to about 95%. the total composition (weight: weight).

Formulations suitable for administration to the eyes include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, such as sucrose and acacia or tragacanth; Pills comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth rinses comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter and a salicylate.

Formulations suitable for intrapulmonary or nasal administration having a particle size, for example between the range of 0.1 to 500 microns (including particle sizes within a range of between 0.1 and 500 microns in increment of microns such as 0.5, 1, 30 microns, 35 microns, etc.), which are administered by rapid inhalation through the nasal passage or by inhalation through the mouth to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be supplied with other therapeutic agents such as the compounds used herein in the treatment or prophylaxis of conditions associated with HIV activity.

Formulations suitable for vaginal administration may be presented as ovules, buffers, creams, gels, pastes, foams or spray formulations, which contain in addition to the active ingredient, carriers such as those known in the art as suitable.

Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the projected recipient's blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations are presented in unit dose or multiple dose containers, for example, sealed vials and flasks and can be stored in a freeze-dried (lyophilized) condition that requires only the addition of the sterile liquid carrier, eg water for injection, immediately before use. Extemporaneous injection solutions or suspensions are prepared from sterile powders, granules, and tablets of the type previously described. Preferred unit dosage formulations are those containing a daily dose, a unit daily sub-dose or a fraction thereof, of the active ingredient. Thus, for example, a daily dose of a salt of the present invention can be provided as a single tablet or in multiple tablets (eg, two or three tablets).

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having questions regarding the type of the formulation, for example suitable for oral administration, may include flavoring agents.

Pharmaceutical formulations that are also within the scope of the present invention provide controlled release of the active ingredient, to allow less frequent dosing to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the present invention also provides pharmaceutical compositions comprising one or more salts of the present invention formulated for sustained or controlled release.

An effective dose of a salt of the present invention depends, for example, on whether the salt is being used prophylactically (a lower dose is usually required compared to the therapeutic use of the same salt), the delivery method and the pharmaceutical formulation, and will be determined by the specialist using conventional dose scale studies. An effective dose can be expected to be from about 0.0001 mg / kg of body weight per day to about 100 mg / kg of body weight per day. Normally, from about 0.01 to about 10 mg / kg of body weight per day. More typically, from about .01 to about 5 mg / kg of body weight per day. More typically from about .05 to about 0.5 mg / kg of body weight per day. For example, the daily dose for a human adult of approximately 70 kg of body weight will normally fluctuate within the range of 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of a multiple dose or dose. By way of example, the dose of a salt of the present invention in a unit dose formulation that will be administered once a day can be from 1 mg to 100 mg, such as from 30 mg to 60 mg, such as a daily dose of 30 mg or a daily dose of 60 mg.

Combination TherapyEach of the citrate, succinate and malonate salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2 , Ethyl 5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate can be used in combination with other therapeutic agents for the treatment or prophylaxis of AIDS and / or one or more other diseases present in a human subject suffering from AIDS (for example, bacterial and / or fungal infections, other viral infections such as hepatitis B or hepatitis C, or cancers such as Kaposi's sarcoma). The additional therapeutic agent (s) can be formulated together with one or more salts of the present invention (eg, formulated together with a tablet).

Examples of such additional therapeutic agents include agents that are effective for the treatment or prophylaxis of viral, parasite or bacterial infections or associated conditions or for the treatment of tumors or related conditions, include 3'-azido-3'-deoxythymidine (zidovudine) , AZT), 2'-deoxy-3'-thiacytidine (3TC), 2 ', 3'-dideoxy-2', 3'-didehydroadenosine (D4A), 2 ', 3'-dideoxy-2 \ 3'- didehydrotimidine (D4T), carbovir (carbocyclic 2 ', 3'-dideoxy-2', 3'-didehydroguanosine), 3'-azido-2 ', 3'-dideoxyuridine, 5-fluorothymidine, (E) -5- (2 -bromovinyl) -2'-deoxyuridine (BVDU), 2-chlorodeoxyadenosine, 2-deoxychoformycin, 5-fluorouracil, 5-fluorouridine, 5-fluoro-2'-deoxyuridine, 5-trifluoromethyl-2'-deoxyuridine, 6-azauridine, 5-fluoroorotic acid, methotrexate, triacetyluridine, 1- (2'-deoxy-2'-fluoro-1-arabinosyl) -5-yodocytidine (FIAC), tetrahydro-imidazo (4,5, 1-Jk) - (1 , 4) -benzodiazepin-2 (1 H) -thione (TIBO), 2'-nor-cyclic GMP, 6-arabinoside of methoxypyrin a (ara-M), 6-methoxypurine 2'-0-valerate arabinoside; cytosine arabinoside (ara-C). 2 ', 3'-dideoxynucleosides such as 2,3'-dideoxycytidine (ddC), 2', 3'-dideoxydenosine (ddA) and 2 ', 3'-dideoxyinosine (ddl); acyclic nucleosides such as acyclovir, penciclovir, famciclovir, ganciclovir, HPMPC, PMEA, PMEG, PMPA, PMPDAP, FPMPA, HPMPA, HPMPDAP, (2R, 5R) -9-tetrahydro-5- (phosphonomethoxy) -2-furanyladenine, (2R , 5R) -1-tetrahydro-5- (phosphonomethoxy) -2-furanylthymidine; other antivirals including ribavirin (adenine arabinoside), 2-thio-6-azauridine, tubercidin, aurintricarboxylic acid, 3-deazancoplanocin, neoplanocin, rimantidine, adamantine, and foscarnel (trisodium phosphonoformate); antibacterial agents including fluoroquinolones bactericins (ciprofloxacin, pefloxacin and the like); bactericidal aminoglycoside antibiotics (streptomycin, gentamicin, amikacin and the like); β-lactamase inhibitors (cephalosporins, penicillins and the like); other antibacterials including tetracycline, isoniazid, rifampin, cefoperazone, claithromycin and azithromycin, antiparasitic or antifungal agents including pentamidine (1,5-bis (4'-aminophenoxy) pentane), 9-deaza-inosine, sulfamethoxazole, sulfadiazine, quinapyramine, quinine, fluconazole, ketoconazole, itraconazole, amphotericin B, 5-fluorocytosine. clotrimazole, hexadecylphosphocholine and nystatin; renal excretion inhibitors such as probenicid; nucleoside transport inhibitors such as dipyridamole, dilazep and nitrobenzylthioinosine, immunomodulators such as FK506, cyclosporin A, thymosin a-1; cytokines including TNF and TGF-β; Interferons including IFN-a, IFN-β, and IFN-α; interleukins including various interleukins, granulocyte / macrophage colony stimulation factors including GM-CSF, G-CSF, M-CSF, cytokine antagonists including anti-TNF antibodies, anti-interleukin antibodies, soluble interleukin receptors, kinase C inhibitors of protein and the like.

Examples of suitable active therapeutic agents or ingredients that can be combined with the salts of the present invention and having activity against HIV, include 1) HIV protease inhibitors, for example, amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, ritonavir, lopinavir + ritonavir, nelfinavir, saquinavir, tipranavir, brecanavir, daranavir, TMC-126, TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), AG1859, DG35, L-756423, RO0334649, KNI-272, DPC-681, DPC-684, and GW640385X, DG 17, PPL-100, 2) a non-nucleoside inhibitor HIV reverse transcriptase for example, capravirin, emivirine, delaviridin, efavirenz, nevirapine, (+) calanolide A, etravirine, GW5634 , DPC-083, DPC-961, DPC-963, MIV-150, and TMC-L20, TMC-278 (rilpivirine), efavirenz, BILR 355 BS, VRX 840773, UK-453.061, RDEA806, 3) a nucleoside inhibitor HIV reverse transcriptase, for example, zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, amdoxovir, vucitabine, alovudine, MTV-210, racivir (-FTC), D-d4FC, emtricitabine, phosphatide, tixodil of fozivudine, tixodil of foslvudine tidoxil, apricitibine (AVX754), amdoxovir. KP-1461, abacavir + lamivudine, abacavir + - lamivudine + zidovudine, zidovudine + lamivudine, 4) an HIV reverse transcriptase nucleotide inhibitor, eg tenofovir, tenofovir disoproxil fumarate + emtricitabine, disodroxil fumarate + emtricitabine - + efavirenz, and adefovir, 5) an inhibitor of HIV integrase, for example, curcumin derivatives of curcumin, quicoric acid, derivatives of quicoric acid, 3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives of aurintricarboxylic acid, phenethyl ester of caffeic acid, phenethyl ester derivatives of caffeic acid, tyrphostin, tyrphostin derivatives, quercetin, quercetin derivatives, S-1360, zintevir (AR-177). L-870812, and L-870810, MK-0518 (raltegravir), BMS-707035, MK-2048, BA-011, BMS-538158, GSK364735C, 6) a gp41 inhibitor, for example, enfuvirtide, sifuvulide, FB006M, TRI -1144, SPC3, DES6, Locus gp41, CovX, and REP 9, 7) a CXCR4 inhibitor, eg, AMD-070, 8) an entry inhibitor, eg, SP01A, TNX-355, 9) a gp120 inhibitor , for example, BMS-488043 and BlockAide / CR, 10) an inhibitor of oxidase G6PD and NADH, for example, immunitin, 11) a CCR5 inhibitor, for example, aplaviroc, vicriviroc, INCB9471, PRO-140, INCB15050, PF- 232798, CCRSmA b004, and maraviroc, 12) an interferon, for example, pegylated rlFN-alpha 2b, pegylated rlFN-alpha 2a, rlFN-alpha 2b, IFN alpha-2b XL, rlFN-alpha 2a, consensus IFN alpha, infergen , rebif, locteron, AVI-005, PEG-infergen, pegylated IFN-beta, oral interferon alfa, feron, reaferon, alpha intermax, r-IFN-beta, infergen + actinmune, IFN-omega with DUROS, and albuferon, 13) ribavirin analogs, eg, copegus, levovirin, VX-497, and viramidine (tarib avirin) 14) NS5a inhibitors, e.g., A-831 and A-689, 15) NS5b polymerase inhibitors, e.g., NM-283, valopicitabine, R1626, PSI-6130 (RL 656), HrV-796, BILB 1941 MK-0608, NM-107, R7128. VCH-759, PF-S68554, GSK625433, and XTL-2125, 16) NS3 protease inhibitors, for example, SCH-503034 (SCH-7), VX-950 (Telaprcvir), IT NI 91 and BILN-2065, 17 ) alpha-glucosidase 1 inhibitors, eg, MX-3253 (celgosivir) and UT-231B, 18) hepatoprotective, for example, IDN-6556, ME3738, MitoQ, and LB-84451, 19) inhibitors without HIV nucleoside, for example, benzimidazole derivatives, benzo-1,2,4-thiadiazine derivatives and phenylalanine derivatives, 20) other drugs for treating HIV, for example, zadaxin, nitazoxanide (aligns), BIVN-401 (virostat), DEBIO- 025, VGX-410C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN-17, KPE02O03002, actilon (CPG-10101), KRN-7000, civacir, GI-5005, ANA-975 (isatoribine), XTL-6865 , ANA 971, NOV-205, tarvacin, EHC-18, and NIM811, 21) pharmacokinetic enhancers, eg, BAS-100 and SPI452, 22) RNAse H inhibitors, eg, ODN-93 and ODN-112, 23 ) other anti-HIV agents, for example, VGV-1, PA-457 (bevirimat), ampligen, HRG214, cytolin, poly mun, VGX-410, KD247, AM2 0026, CYT 99007. A-221 HIV, BAY 50-4798, MDX010 (iplimumab), PBS119, ALG889 and PA-1050040.

Again by way of example, the following list describes exemplary HIV antivirals with their corresponding US Patent numbers which may be combined with the salts of the present invention.

HIV Antivirals of Example v Patent NumbersZiagen (Abacavir Sulfate, Patent Publication No. 5,034,394)Epzicom (Abacavir sulfate / lamivudine, Patent Publication No. 5,034,394)Hepsera (Adefovir dipivoxil, Patent PublicationNo. 4,724,233)Agenerase (Amprenavir- Patent Publication No. 5,646,180)Reyataz (Atazanavir Sulfate, Patent Publication No. 5,849,911)Rescriptor (Delavirdine Mesylate, Patent Publication No. 5,563,142)Hivid (Dideoxycytidine; Zalcitabine, Patent Publication No. 5,028,595)Videx (Didesoxiihosina; Didanosine, Publication ofPatent No. 4,861, 759)Sustiva (Efavirenz, Patent Publication No. 5,519,021)Emtriva (Emtricitabine, Patent Publication No. 6,642,245)Lexiva (Fosamprenavir Calcium, Patent PublicationUS 6,436,989)Virudin, Triapten; Foscavir (Sodium of foscarnet, Patent Publication US 6,476,009)Crixivan (Indinavir Sulfate, Patent Publication US 5,413,999)Epivir (Lamivudine, Patent Publication US 5 047,407)Combivir (Lamivudine / Zidovudine, US Patent Publication 4,724,232)Aluviran (Lopinavir)Kaletra (Lopinavir / ritonavir, US Patent Publication5,541, 206)Viracept (Nelfinavir mesylate, US Patent Publication 5,484,926)Viramune (Nevirapine, US Patent Publication 5,366,972)Norvir (Ritonavir, US Patent Publication 5,541,206)Invite; Fortovase (saquinavir mesylate, US Patent Publication 5,196,438)Zerit (Stavudine, US Patent Publication 4,978,655) Truvada (disoproxil fumarate of tenofovir / emtricitabine, US Patent Publication 5,210,085)Aptivus (Tipranavir)Retrovir (Zidovudine; Azidothymidine, US Patent Publication 4,724,232)HIV inhibition methodsIn a further aspect, the present invention provides methods for treating Acquired Immune Deficiency Syndrome (AIDS), wherein each method includes the step of administering to a human suffering from AIDS, a therapeutically effective amount of a salt of the present invention. , or a hydrate of a salt of the present invention. AIDS treatment includes the reduction of at least one symptom of AIDS, and / or decreasing or preventing the progression of the disease. Typically the therapeutically effective amount of the salt is administered to a human in the form of a pharmaceutical composition as described in the "pharmaceutical compositions" section. Typically, the pharmaceutical composition is administered orally, for example in the form of a tablet. Examples of therapeutically effective daily doses of one or more salts of the present invention, or hydrates thereof, are from 1 mg to 100 mg such as from 10 mg to 30 mg. The salts of the present invention can be administered daily, for example in the form of one or more tablets that include an amount of salt that provides an effective amount such as 10 mg or 30 mg or 60 mg, of the free base when the salt it dissociates in an aqueous medium inside the human body.

Administration RoutesOne or more salts of the present invention are administered through any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It can be appreciated that the preferred route may vary, for example, with the condition of the receiver. An advantage of the salts of the present invention is that they are orally bioavailable and can be dosed orally.

Examples and Sample Modalities:See also International Publication WO 2006/110157, the disclosure of which is incorporated in its entirety by reference to the present invention, particularly pages 167 to 174. Example 1. Synthesis of N - [(S) ( { [(2R, 5R ) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate ethyl.2-deoxy-2-f luoro-3,5-di-0-benzoyl-a-D-arabinofuranosylbromide (2)The compound (2) was synthesized according to the synthetic schemes described in Tann and Associates Publication, JOC, 1985, Vol. 50 page 3644 and Howell and associates, JOC, 1988, Vol. 53, page 85.

To a solution of 1.3.5-tri-0-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (1) (120 g, 258 mmol), commercially available in Davos or CMS Chemicals, in CH 2 Cl 2 (1 L) , HBr / 33% acetic acid (80 mL) was added. The mixture was stirred at room temperature for 16 hours, cooled with ice-water and neutralized slowly for 1 to 2 hours with NaHCO 3 (150 g / 1.5 L of solution).

The CH2Cl2 phase was separated and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHCO 3 until there was no acid. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to provide product 2 in the form of a yellow oil (-115 g).2-deoxy-2-f luora-3,5-di-0-benzoyl-p-D-arabinofu ra nos i 1-9 H -6- chloropurine (3)The compound (3) was synthesized according to the synthetic schemes described in Ma and Associates Publications, J. Med. Chem., 1997, Vol. 40, page 2750; Márquez et al., J. Med. Chem., 1990, Vol. 33, page 978; Hildebrand and associates, J. Org. Chem., 1992, Vol. 57, page 1808 and Kazimierczuk and associates, JACS, 1984, Vol. 106, page 6379.

To a suspension of NaH (14 g, 60% in Acetonitrile (900 ml_), 6-chloropurine (52.6 g) was added in 3 portions, the mixture was stirred at room temperature for 1.5 hours. solution of 2 (258 mmol) in Acetonitrile (300 ml_) The resulting mixture was stirred at room temperature for 16 hours The reaction was quenched with acetic acid (3.5 mL), filtered and concentrated under reduced pressure. The mixture was partitioned between CH2Cl2 and water, the organic phase was dried over MgSO4, filtered and concentrated, the residue was treated with CH2Cl2 and then EtOH (generally -1: 2) to precipitate the desired product 3 in the form of a yellowish solid (83). g, 65% of 1).2-deoxy-2-fluoro-p-D-arabinofuranosyl-6-methoxyadenine (4)To a suspension of 3 (83 g, 167 mmol) in methanol (1 L) at a temperature of 0 ° C, NaOMe (25% by weight, 76 ml_) was added. The mixture was stirred at room temperature for 2 hours, and then quenched with acetic acid (~ 11 mL, pH) 7). The mixture was concentrated under reduced pressure and the resulting residue was partitioned between hexane and water (approximately 500 mL of hexane and 300 mL of water). The aqueous layer was separated and the organic layer was mixed once more with water (approximately 300 mL). The water fractions were combined and concentrated under reduced pressure at -100 mL. The product, 4, was precipitated and collected by filtration (42 g, 88%).2-deoxy-2-fluoro-5-carboxy-p-D-arabinofuranosyl-6Methoxiadenine (5)The compound (5) was synthesized according to the synthetic schemes described in Moss and Associates Publication, J. Chem. Soc, 1963, page 1149.

A mixture of Pt / C (10%, 15 g (20-30 mol% equiv.) In the form of a water paste) and NaHCO 3 (1.5 g, 17.94 mmoles) in H20 (500 ml_), was stirred to a temperature of 65 ° C under H2 for 0.5 hours. The reaction mixture was allowed to cool subsequently, placed under a vacuum and rinsed several times with N2 to completely remove all of the H2. Compound 4 (5.1 g, 17.94 mmol) was subsequently added at room temperature. The reaction mixture was stirred at a temperature of 65 ° C under 02 (balloon) until the reaction was completed by LC-MS (usually 24 to 72 hours). The mixture was cooled to room temperature and filtered. The Pt / C was washed extensively with H20. The combined filtrates were concentrated to ~30 mL, and acidified (pH 4) through the addition of HCI (4N) at a temperature of 0 ° C. A black solid precipitated which was collected by filtration. The crude product was dissolved in a minimum amount of methanol and filtered through a pad of silica gel (eluting with methanol). The filtrate was concentrated and crystallized from water to provide compound 5 (2.5 g) in the form of a cream colored solid.< 6 >(2'R, 3'R, 4'R, 5, R) -6-Methoxy-9- [tetrahydro-4-iodo-3-f luoro-5- (diethoxyphosphinyl) methoxy-2-furanyl] purine (6)The compound (6) was synthesized according to synthetic schemes described in Zemlicka et al., J. Amer. Chem., Soc, 1972, Vol. 94, page 3213.

To a solution of 5 (22 g, 73.77 mmol) in DMF (400 mL), DMine dineopentyl acetal (150 mL, 538 mmol) and methanesulfonic acid (9.5 mL, 146.6 mmol) were added. The reaction mixture was stirred at a temperature of 80 to 93 ° C (internal temperature) for 30 minutes, then cooled to room temperature and concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic phase was separated and washed with NaHCO 3, followed by brine, dried over MgSO 4, filtered and concentrated under reduced pressure. The diethyl e (hydroxymethyl) phosphonate residue (33 mL, 225 mmol) in CH2Cl2 (250 mL) was dissolved and cooled to -40 ° C. A solution of iodine monobromide (30.5 g, 1.1 mol) in CH2Cl2 (100 mL) was added as drops. The mixture was stirred at a temperature of -20 to -5 ° C for 6 hours.

Subsequently the reaction was quenched with NaHCO3 and Na2S203. The organic phase was separated and the water phase was extracted with CH2Cl2. The combined organic phases were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to provide product 6 (6 g, 15.3%).

Alternative Procedure for the Preparation of 6A solution of 5 (2.0 g, 6.7 mmol) in THF (45 mL) was treated with triphenyl phosphine (2.3 g, 8.7 mmol) under N2. Diisopropyl azodicarboxylate (1.8 g, 8.7 mmol) was added slowly. The resulting mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure to dryness. The residue was dissolved in CH2Cl2 (20 mL) and subsequently treated with diethyl (hydroxymethyl) phosphonate (4.5 g, 27 mmol). The mixture was cooled to a temperature of -60 ° C and then a cold solution of 2 g iodine monobromide, 9.6 mmol) in CH2Cl2 (10 mL) was added. The reaction mixture was warmed to a temperature of -10 ° C and subsequently maintained at a temperature of -10 ° C for 1 hour. The reaction mixture was diluted with CH2Cl2, washed with saturated aqueous NaHCO3, and subsequently with aqueous sodium thiosulfate. The organic phase was separated, dried over MgSO4 and concentrated under reduced pressure to dryness. The reaction mixture was purified by silica gel chromatography (eluting with 25% ethyl acetate in CH 2 Cl 2, then switching to 3% methanol in CH 2 Cl 2) to yield product 6 (0.9 g, 33%).(2, R, 5, R) -6-Methoxy-9- [3-fluoro-2,5-dihydro-5- (diethoxyphosphinyl) methoxy-2-furanyl] purine (7)To a solution of compound 6 (6 g, 11.3 mmol) in acetic acid (2.5 ml_) and methanol (50 ml_), NaCl (10-13%) (50 ml) was added dropwise. The reaction mixture was subsequently stirred for 0.5 hours and concentrated under reduced pressure. The residue was treated with ethyl acetate and subsequently filtered to remove the solids. The filtrate was concentrated and the residue was purified by silica gel chromatography to provide product 7 (4 g, 88%).

Disodium salt of (2'R, 5"R) -9- { 3-f luoro-2,5-di h id ro-5- f osphonomethoxy-2-f uranyl) adenine (8)A solution of compound 7 (2.3 g, 5.7 mmol) in methanol (6 mL) was mixed with aluminum hydroxide (28-30%) (60 mL). The resulting mixture was stirred at a temperature of 120 ° C for 4 hours, cooled and then concentrated under reduced pressure. The residue was dried under vacuum for 12 hours. The residue was dissolved in DMF (40 mL) and bromotrimethylsilane (3.5 mL) was added. The mixture was stirred at room temperature for 16 hours, and then concentrated under reduced pressure. The residue was dissolved in aqueous NaHC03 (2.3 g in 100 mL of water). The solution was evaporated and the residue was purified on a C-18 column (40 pM), eluting with water. The aqueous fractions were freeze-dried to provide the disodium salt 8 (1.22 g, 57%).

Example of Preparation of Monoamidate (9)Disodium salt 8 (25 mg, 0.066 mmol), aster (S) -Ala-O-cyclobutyl hydrochloride (24 mg, 2 eq. 0.133 mmole) and phenol (31 mg, 0.333 mmol) were mixed in anhydrous pyridine ( 1 mL). Triethylamine (111 pL, 0.799 mmol) was added and the resulting mixture was stirred at a temperature of 60 ° C under nitrogen. In a separate flask, 2 '- Al dri ti or I (122 mg, 0.466 mmol) and triphenylphosphine (103 mg, 0.466 mmol) were dissolved in anhydrous pyridine (0.5 ml_) and the resulting yellow solution was stirred for 15 to 20 minutes. minutes Subsequently the solution was added to the solution of 8 in one portion. The combined mixture was stirred at a temperature of 60 ° C under nitrogen for 16 hours to provide a solution with light yellow to light brown color. Subsequently, the mixture was concentrated under reduced pressure. The resulting oil was dissolved in CH2Cl2 and purified by silica gel chromatography (eluting with a linear gradient of 0 to 5% MeOH in CH2Cl2) to provide an agent. The resulting oil was dissolved in acetonitrile and water and purified by preparative HPLC (linear gradient, 5 to 95% acetonitrile in water). The pure fractions were combined and freeze-dried to provide the mono amidate 9 in the form of a white powder.

Example of Preparation of bis amidate (10)Disodium salt 8 (12 mg, 0.032 mmol) and ester hydrochloride (S) -Ala-O-n-Pr (32 mg, 6 eq., 0.192) were mixed.mmoles) in anhydrous pyridine (1 ml_). Triethylamine (53 [mu] -, 0.384 mmol) was added and the resulting mixture was stirred at a temperature of 60 [deg.] C. under nitrogen. In a separate flask, 2 '-Al d riti or I (59 mg, 0.224 mmol) and triphenylphosphine (49 mg, 0.224 mmol) were dissolved in anhydrous pyridine (0.5 ml_) and the resulting yellow solution was stirred for 15 to 20 minutes. . Subsequently the solution was added to the solution of 8 in one portion. The combined mixture was stirred at a temperature of 60 ° C under nitrogen for 16 hours to provide a light yellow to light brown solution. Subsequently, the mixture was concentrated under reduced pressure. The resulting oil was dissolved in CH2Cl2 and purified by silica gel chromatography (eluting with a linear gradient of 0 to 5% MeOH in CH2Cl2) to provide an oil. The resulting oil was dissolved in acetonitrile and water and purified by preparative HPLC (linear gradient, 5 to 95% acetonitrile in water). The pure fractions were combined and freeze-dried to provide bis-amidate in the form of a white powder.

Example of Preparation of Monoamidate (11)Compound 8 (1.5 g, 4 mmol) was mixed with HCl salt of ethylalanine ester (1.23 g, 8 mmol) and phenol (1.88 g, 20 mmol). Anhydrous pyridine (35 ml_) was added followed by TEA (6.7 ml_, 48 mmol). The mixture was stirred at a temperature of 60 ° C under nitrogen for 15 to 20 minutes. 2'-Aldrithiol (7.3 g) was mixed in a separate flask with triphenylphosphine (6.2 g) in anhydrous pyridine (5 ml) and the resulting mixture was stirred for 10 to 15 minutes. to provide a light yellow, lightened solution. Subsequently, the solution was added to the previous mixture and stirred overnight at a temperature of 60 ° C. The mixture was concentrated under reduced pressure to remove the pyridine. The resulting residue was dissolved in ethyl acetate and washed with a saturated sodium bicarbonate solution (2x) and subsequently with a saturated sodium chloride solution. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The resulting oil was dissolved in dichloromethane and loaded onto a dry CombiFlash column, 40 g, eluting with a linear gradient of 0 to 5% methanol in dichloromethane for 10 minutes and then in 5% methanol in dichloromethane for 7 to 10 minutes. . The fractions containing the desired product were combined and concentrated under reduced pressure to provide a foam. The foam was dissolved in acetonitrile and purified by preparative HPLC to provide 11 (0.95 g).11 (950 mg) was dissolved in a small amount of acetonitrile and allowed to settle at room temperature overnight. The solid was collected by filtration and washed with a small amount of acetonitrile. The filtrate was reduced under vacuum and then loaded onto a Chiralpak AS-H column equilibrated in buffer A, 2% ethanol in acetonitrile. Isomer A, 12, was eluted with buffer A at 10 mL / minute for 17 minutes. After this, buffer B, 50% methanol in acetonitrile, was used to elute isomer B, 13, separately from the column in 8 minutes.

All the solvent was removed and subsequently re-dissolved separately in acetonitrile and water. The samples were separately frozen (mass - 348 mg). The 12-isomer is shown below.

Example 2. Classification of the salt forms of N - ((S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5 ethyl dihydrofuran-2-yl] oxy} oxymethyl) phenoxyphosphino] -L-alaninate The following acids were classified to determine whether they formed suitable crystalline salts of N - [(S) ( { [(2R, 5R)] -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy}.methyl) phenoxyphosphinoyl] -L-alaninate: mineral acids (HX, where X = halogen; H3P04); organic sulfonic acids (RSO3H, where R = Me, Et, Ph, (+) -canfor-10-acidsulfonic; Naphthalene-2-sulfonic acid; naphthalene-1, 5-disulfonic acid); monocarboxylic acids (RCO2H, wherein R = H, Me, Et, Ph, rrans-PhCH = CH, CI2CH, PhCONHCH2), and dicarboxylic acids (malonic, succinic, fumaric, adipic, oxalic, maleic).

Solids were obtained with three of the above acids when mixed with N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro -2,5-dihydro-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate: trifluoroacetic acid, malonic acid and succinic acid. Trifluoroacetic acid is not considered to be pharmaceutically acceptable.

The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphino] -L-alaninate is a prodrug of amidate which passes by decomposition in protic solvents under acidic or basic conditions. For this reason, acids with potentially nucleophilic portions were not included in the initial return of the classification. Subsequently, citric acid, glycolic acid, (S) - (+) - lactic acid, salicylic acid, (S) - (-) - malic acid, (S) - (+) - mandelic acid and ( S) - (+) -glutamic. The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphino] -L-alaninate unexpectedly formed a stable crystalline salt with citric acid. This was an unexpected result because the citric acid includes a hydroxyl group which can act as a nucleophile towards the amidate portion of N - [(S) ( { [(2R, 5R) -5- (6- amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate and potentially pass through a reaction there, in where a covalent bond is formed between N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran -2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate and the hydroxyl group of citric acid with the expulsion of either phenol or alanine ethyl ester.

Example 3. Synthesis of malonate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5- dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninateThe free base form of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2 ethyl] oxy] ethyl) phenoxyphosphino] -L-alaninate was dissolved in warm 2-butanone (15 parts), and malonic acid (0.26 parts) was added to form a solution with stirring. Heptane (5 parts) was added to the solution which was slowly cooled to a temperature of about 5 ° C, collected and rinsed with cold 2-butanone / heptane.The malonate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-M) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate from ethyl acetate, was produced in this way with a yield ofapproximately 80%.

The NMR spectrum of the malonate salt had the following characteristics: 1 H NMR (400 MHz, CHLOROFORMO-d) ppm = 8.27 (s, 1 H), 8.26 (s, 1 H), 7.32 - 7.20 (m, 3 H), 7.15 (d.J = 7.8 Hz, 2 H), 6.78 (m, 1 H), 5.88 (br. S., 1 H), 5.78 (s, 1 H), 4.17 (m 2 H), 4.15 - 4.07 (m, 3 H), 3.85 (dd, J = 8.0, 8.0, 1 H), 3.38 (s, 2 H), 1.31 (d, J = 7.0 Hz, 3 H), 1.23 (t, J = 7.0 Hz, 3 H) 31P NMR (162 Hz, CHLOROFORMO-d) ppm = 20.64 (s) 19F NMR (376 MHz, ACETONITRILO-d3) ppm = -335.19 (s) Example 4. Synthesis of N- succinate salt [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl. ) phenoxyphosphonyl] -L-alaninate ethyl.

The free base form of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2 ethyl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate was dissolved in warm 2-butanone (15 parts), and succinic acid (0.28 parts) was added to form a solution with stirring. The solution was cooled slowly to a temperature of about 5 ° C, collected and rinsed with cold 2-butanone, to thereby produce the succinate salt of N - [(S) ( { [(2, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy]. Methyl) phenoxyphosphinoyl] -L-alaninate with a yield of 80% .

The NMR spectrum of the succinate salt had the following characteristics: 1H NMR (400MHz, ACETONITRILO-d3) ppm =8. 26 (s, 1H), 8.15 (s, 1 H), 7.29 (dd, J = 7.6, 7.6 Hz, 2 H). 7.16 (d, 7 = 7.6 Hz, 1 H), 7.12 (d, J = 8.0 Hz, 2 H), 6.78 (m, 1 H), 6.17 (br s, 1 H), 5.89 (m, 2 H), 4.12 - 3.95 (overlapping multiples, 6 H), 2.53 (s, 4 H), 1.24 (d, J = 6.8, 3 H), 1.18 (t, J = 7.2, 3 H).31P NMR (162 MHz, Acetonitrile-d3) ppm = 21.60 (s)19 F NMR (376 MHz, Acetonitrile-d 3) ppm = -135.19 (s)Example 5. Synthesis of Citrate Salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5- dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

The free base form of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2 ethyl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate(about 30 g) was dissolved in hot acetonitrile (16 parts) and citric acid (0.38 parts) was added with stirring. The resulting solution was cooled slowly to a temperature of about 5 ° C, collected and rinsed with cold acetonitrile, and dried, to thereby produce the citrate salt of N - [(S) ( { [(2R , 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate with a yield of approximately 84%.

The NMR spectrum of the citrate salt had the following characteristics: 1 H NMR (400 MHz, DMSO-d 6) ppm = 8.20 (s, 2 H), 7.45 (2 H, br s), 7.29 (dd, J = 7.6, 7.6 Hz, 2 H), 7.13 (dd, J = 7.2 Hz, J = 7.2 Hz, 1 H), 7.12 (dd, J = 8.0 Hz, J = 8.0 Hz 2 H), 6.86 (d, J = 2.4 Hz, 1 H ), 6.14 (s, 1H). 5.97 (d, J = 3.6 Hz, 1H), 5.78 (dd, J = 12.2, 10.4 Hz, 1H), 4.05 (m, 1H), 4.02 (m, 2H), 3.98 (m, 1H), 3.89 (m , 1H), 2.76 (d, J = 15.6 Hz, 2H), 2.66 (d, J = 15.6 Hz, 2H), 1.16 (d, J = 7.2 Hz, 1H).3 P NMR (162 MHz, DMSO-d6) ppm = 22.29 (s)19 F NMR (376 MHz, ACETONYRTHYL-d3) ppm = -133.88 (s) HRMS: m / z. 507.1561; Calculated for C2i H24FN606P: 507.1557.

Anal. Calculated for C21 H24FN606P: C: 46.42; H: 4.62; N: 12.03; P: 4.43; F: 2.72; Found: C: 45.83; H: 4.74; N: 11.81: P: 4.45; F: 2.80.

Example 6. Physicochemical Properties of the Salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5- dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate Representative batches of malonate, succinate and citrate salts of N - [(S) ( { [(2R, 5R) - 5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dih id cleaved n-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate. The melting points of these salts were determined and served as a rigorous measure of stability with a higher melting point indicated a higher level of stability. As shown in Table 1, the citrate salt had the highest melting point. In addition, the heat of fusion (AH (fusion)) of each of the three salts is shown in Table 1. The citrate salt had the highest heat of fusion, indicating a higher degree of solid state crystallinity than the other two salts.

Table 1. Melting Temperature and Melting Heat of Succinate Salts, Malonate and Citrate of N - [(S) ( { [(2R, 5R) -5- (6-ainino-9H-purin-9-il Ethyl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninateThe free base form of N - [(S) ( { [ { 2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran -2-il] oxy} methyl) phenoxyphosphino] -L-alaninate is amorphous, hygroscopic and chemically unstable when stored under open conditions at a temperature of 40 ° C and a relative humidity of 75% (RH). As shown in Table 2, the corresponding succinate, malonate and citrate salts were not hygroscopic at room temperature when exposed to a relative humidity of 92% for several days.

Table 2. Hygroscopicity of Solid States of the Salt Forms of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro -2,5- dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate ethyl aRoom temperatureThe chemical stability of the solid state of the salts of succinate, malonate and citrate of free base, of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9- il) -4-fluoro-2,5-dihydrofuran-2-yl] oxy) methyl) phenoxyphosphinoyl] -L-alaninate were checked under open conditions at a temperature of 40 ° C and a relative humidity of 75% . As shown in Table 3, the citrate salt showed superior chemical stability compared to the citrate and malonate salts.

Table 3. Solid State Stability of the Free Base Form of N ^ - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-il) -4- fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate, and the corresponding salts of Succinate, Citrate and Malonate in(Temperature conditions of 40 ° C / Relative Humidity of 75%Under Open ConditionsOtherFree Base TimeForm impurities(days) (%)(%)0 99.01 0.997 82.95 17.05Free Base14 66.89 33.1122 55.90 44.100 98.95 1.059 95.15 4.85Get ouf of16 92.47 7.53Succinate22 90.43 9.5730 82.92 14.080 97.82 2.189 94.66 5.34Malonato salt 16 92.97 7.0322 93.48 6.5230 85.84 14.160 98.00 2.004 97.27 2.73Citrate salt 12 97.20 2.8017 95.86 4.1427 94.59 5.41Example 7. Composition of Tablets Providing the Equivalent to 10 mg and 30 mg of the free base of N - [(S) ( { [ { 2R, 5R) -5- (6-amino-9H-purin -9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl The citrate salt of N - [(S) ( { [ { 2R, 5R) -5- (6-amino-9H-purin-9-yl) - 4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate was formulated in 10 mg and 30 mg tablets using a roller compaction process. The active ingredient, lactose anhydrous, microcrystalline cellulose and sodium of croscarmellose were first combined, and the mixture was subsequently lubricated with one third of the total amount of magnesium stearate, subsequently compacted with a roller, followed by grinding. The resulting granules were lubricated with the remaining amount of magnesium stearate and pressed into tablets.

Table 4 shows the composition of the tablets that include the citrate salt of N - [(S). { . { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate, and which provides either 10 mg or 30 mg of the free base form of the compound, when the citrate salt dissociates in an aqueous medium.

Table 4a Equivalent of 10% 2/2 of the free base form of the compound. The actual weight of the drug substance will be adjusted to take into account the purity of the substance.b Equivalent to 10 mg of the free base form of the compound.c Equivalent to 10 mg of the free base form of the compound.d The amount of adjusted drug substance will be subtracted from the amount of anhydrous lactose.8 The abbreviation NF, means national form, and the abbreviation HSE, means Reference of Local Compendium, which is an internal standard used in Gilead SciencesExample 8Comparison of Active Metabolite Lymphocyte Load After Administration to N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro Lymphocytes -2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate (Prodrug) or Source Compound.

The N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-d-hydrofuran-2- Ethyl] oxy] phenoxyphosphinoyl] -L-alaninate isa prodrug that is hydrolyzed in the human body to produce a hydrolysis product which will hereinafter be referred to as "the parent compound". The parent compound is phosphorylated within the human body to produce a biologically active phosphorylated product (hereinafter "the active metabolite") that inhibits the activity of the reverse transcriptase enzyme.

To characterize the intracellular metabolism of the drug and compound of origin, lymphocyte cells were treated with either 1 μ? of prodrug or 100 μ? of origin compound during 2, 6, and 24 hours. Peripheral blood mononuclear cells (PBMCs) were isolated from human fluffy coatings (Stanford Blood Bank, Palo Alto, CA) using centrifugation in Ficoll Paque Plus (GE Healtcare, Piscataway, NJ) according to the manufacturer's procedure. Isolated PBMCs from 3 to 4 independent donors were maintained in RPMI-1640 medium with 20% fetal bovine serum and antibiotics (inactive state) or activated in the presence of interleukin 2 (20 units / ml_, Roche Biochemicals, I ndianapolis, IN) and phytohemagglutinin PHA-P (1 pg / mL, Sigma) for 3 to 4 days before starting the experiments.

Human CCRF-CEM T cells transformed in the American Type Culture Collection (Manassas, VA) were obtained and cultured in an RPMI-1640 medium supplemented with 10% FBS and antibiotics. An aliquot of cells (2-3x106 cells) was collected at each time point, counted, pelleted by centrifugation, resuspended in 0.5 mL of the original treatment medium and made in layers in 0.5 mL of Nyosil 25 oil. Samples were rotated in a microcentrifuge for 20 seconds at maximum speed (approximately 800 xg). The top layer of the medium was removed and the oil layer was washed twice with 0.8 mL of phosphate buffered saline. The wash buffer and the oil layer were carefully removed, the cell pellet was resuspended in 0.5 mL of 70% methanol and incubated overnight at a temperature of -70 ° C to facilitate cell lysis. The used cells were centrifuged, the supernatants were collected, dried by vacuum and resuspended in 10 pL of tetrabutyl ammonium acetate containing [5- (6-Amino-purin-9-yl.). 2,5-dihydro-furan-2-yloxymethyl] -phosphonic acid (the non-fluorinated analog of the active metabolite) in the form of an internal standard.

High performance liquid ion-pair-pairing chromatography coupled to positive ion electrode tandem mass spectrometry (LC / MS / MS) was used to quantify intracellular nucleotides. The methods were adapted from those described for the nucleic acid analogue of acyclic phosphonate, adefovir its phosphorylated metabolites and natural nucleotides (Vela, .1.E. and associates, simultaneous quantification of the nucleotide analogue adefovir, its phosphorylated anabolites and triphosphate 2'-deoxyadenosine by LC / MS / MS ion pairing, Journal of Chromatography B Anal., Biomed Technique, Life Sci., Vol 848, 2007, pp 335-343). Standard curves and quality control samples were generated for all materials for analysis using extracts from untreated cells. The standard seven-point curves generally ranged from 0.03 to 20 pmol / million cells and had a linearity in excess of 0.99 for all materials for analysis: The lower quantification limits for all materials for analysis ranged from 0.05 to 0.1 pmol / million cells. Quality control samples of high and low concentration (usually 0.2 and 10 pmol / million cells, respectively) were run with each material for analysis at the start and end of each run to ensure accuracy and precision within 20%.

The compound of origin was incubator in a concentration greater than 100 times (100 μ?) Than the prodrug (1 μ) to facilitate the precise analysis of the accumulation of intracellular metabolites much lower observed after the incubation of lymphocytes with the compound of origin . As shown in Table 5, the prodrug induced 76-, 290- and 140-fold increase levels of the active metabolite relative to the parent compound after incubation with CEM-CCRF, inactive PBMCs and activated PBMCs, respectively. The levels of active metabolite were normalized based on the extracellular concentration after incubations with 1 μ? of prodrug or with 100 μ? of the origin compound.

Table 5Example 9Comparison of Anti-HIV Activity of the Prodrug and the Compound of OriginThe terms "prodrug" and "origin compound" have the meanings set forth in Example 8.

MT-2 cells were maintained in RPMI-1640 medium supplemented with antibiotics and 10% fetal bovine serum (FBS). MT-2 cells were infected with HIV-1 IIIB at a multiplicity of infection (moi) of 0.01 and were added in 96-well plates with serial dilutions and the compounds were tested at a density of 20,000 cells / reservoir. After incubation for 5 days, the virus-induced cytopathic effect was determined using a CelITiter-Glo ™ cell viability assay (Promega, Madison, Wl) and expressed as a percentage of the signal from the samples with the virus replicate completely suppressed after of the signal subtraction of the untreated control. The concentration of each drug that inhibited the cytopathic effect induced by virus by 50% (EC5o), was determined by non-linear regression. The activity against mutants resistant to NRT1 was determined in parallel with the wild-type control virus and the change in doubles in EC50 was calculated.

Human peripheral blood mononuclear cells (PBMCs) were isolated from sponge donor coatings using centrifugation in Ficoll Paque Plus and activated for 4 to 5 days in RPMI-1640 medium with 20% FBS, antibiotics, interleukin-2 (20 units / mL) and phytohemagglutinin PMA-P (2 pg / mL). PBMC activated with HIV-1 BaL were infected for 3 hours, washed, plated in 96-well plates (250,000 cells / tanks) and incubated with serial dilutions of tested compounds for 5 days, at which point they were harvested. the cellular supernatants and virus production was determined using commercial HIV-1 p24 ELISA (Beckman Coulter, Miami, FL). The concentration of each drug that inhibits p24 antigen production by 50% (EC50) was determined by regression analysis.

The effect of the addition of the pro-portions of the anti-HIV activity was evaluated in stimulated MT-2 and PBMC infected with HIV-1. As shown in Table 6, the prodrug was 71- and 2,300-fold more potent than the MT-2 origin compound and activated PBMC, respectively.

Table 6. Anti-HIV activity of the prodrug and compound of origin in a cell line derived from lymphoid and primary lymphoid cells.

Example 10Oral Bioavailability of the Citrate Salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5 -dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate. After the. Administration in the Form of Tablets to Beagle DogsThe dosing group consisted of 3 non-na'fve male Beagle dogs. The animals were fasted overnight before the administration of the dose and up to 4 hours after dosing. The dogs were administered a single tablet containing 41.38 mg of the citrate salt of the prodrug (which provides 30 mg of the prodrug per tablet).

The tablet consisted of 13.79% of the prodrug citrate salt, 66% of anhydrous lactose, 15.21% of microcrystalline cellulose, 3.5% of croscarmellose sodium and 1.5% of magnesium stearate on a weight basis by weight. Plasma samples were obtained before dosing (0 hours) and at 0.083, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 12, 24 hours. Blood samples were collected in Vacutainer ™ tubes containing EDTA-K3. The blood samples were centrifuged at a temperature of 4 ° C to separate the plasma. A 100 μ aliquot was first diluted? of each plasma sample with 300 μ? 80% acetonitrile / water containing 200 nM internal standard. After centrifugation of the protein precipitate, 100 μ? of the supernatant and used for analysis. Standard curves and quality control samples were prepared in dog plasma, from animals that were not dosed with the prodrug. The samples were analyzed through a partially validated liquid chromatography coupling method with triple quadrupole mass spectrometry.

Administration of the citrate salt of the prodrug in the form of a tablet resulted in rapid absorption of the prodrug and the parent compound derived therefrom. As summarized in table 7, after administration, exposure of the plasma to the prodrug and the parent compound was observed. The oral bioavailability of the intact prodrug was 11.4%. These results were not markedly different from those observed after oral administration of the prodrug in a solution formulation, illustrating the effectiveness of the tablets containing the citrate salt of the prodrug to deliver the prodrug and its metabolites into the systemic circulation.

Table 7. Average Plasma Pharmacokinetic Parameters for the Prodrug and the Source Compound after Oral Administration of a Prodrug Citrate Salt Tablet Formulation as an Average Dose of 3.05 mg / kg equivalents (average, n = 3).

Example 11Differential Scanning Calorimetry (DSC) of Citrate Salts, Malonate and N-Succinate [(S) ( { [ { 2R: 5R) -5- (6-amino-9H-purin-9-il) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate.

The differential scanning calorimetry accurately measures the temperatures and heat flux associated with thermal transitions in a material. The DSC traces of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran- salts. Ethyl 2-yl] oxy] methyl) phenoxyphosphino] -L-alaninate of the present invention were generated using a TA Instrument (New Castie, DE) DSC 2010 in a scanning range of 5 ° C min. Figure 1 shows the characteristic DSC trace of the malonate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4- fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate ethyl The DSC thermogram revealed a simple endotherm corresponding to the melting point of (120.43 ° C, AHf = 73.72 J / g) followed by an exotherm whenever the decomposition of the malonate salt corresponds.

Figure 2 shows the characteristic DSC trace of the succinate salt of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4- fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate. The DSC thermogram of the succinate salt of GS-913, revealed a single endotherm corresponding to the melting point (137.44 ° C, AHf = 66.18 J / g) followed by a single exotherm corresponding to the decomposition of the salt.

Figure 3 shows the characteristic DSC trace for the citrate salt of N - [(S) ( { [2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro -2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphino] l -L-alaninate. The DSC thermogram of the citrate salt revealed a single endotherm corresponding to the melting point (149.41 ° C, AHf = 85.72 J / g) followed by a single exotherm corresponding to the decomposition of the citrate salt.

The citrate salt has a significantly higher heat of fusion than the malonate and succinate salts, indicating a higher degree of solid state crystallinity.

Example 12X-Ray Diffraction Analysis (XRD) of Citrate, Malonate and Succinate Salts of N - [(S) ( { [(2R, 5R) -5- (6-amino-9H-purin-9-il) Ethyl 4-fluoro-2,5-dihydrofuran-2-yl] oxy] methyl) phenoxyphosphinoyl] -L-alaninate.

The X-ray powder diffraction patterns (XRPD) of the N - [(S) salts. { [(2R, 5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-2,5-dihydrofuran-2-yl] oxy} methyl) phenoxyphosphinoyl] -L-alaninate of the present invention, were generated using two methods. In the first method, a Shimadzu XRD 6000 instrument was used with the following attributes: X-ray tube Cu, 2.2KW, NF (normal focus); Curved graphite monochromator; vertical goniometer, radius 185 mm; divergence slots: 0.5 °, 1.0 °, 0.05 mm; Soller slots: 0.05 °, 1st, 2nd; Reception slots: 0.15mm, 0.3mm; scintillation detector (Nal) HV 500-1200.

In the second method, a Shimadzu XRD 6000 instrument was used with the following attributes: Cu X-ray tubes, 35 kv, 40 ma current; continuous scan, monochromator, divergence slot, 1st: Soller slot 1o, reception slot 0.3 mm.

The XRPD data for the malonate and succinate salts were obtained using method 1. The XRPD data for the citrate salt were obtained using method 2.

It will be understood that experimental deviations may slightly change the information of the absorption band XRPD (peak). Therefore, the numbers reported in this patent resulting from the XRPD patterns of the salts of the present invention, will be the same or essentially the same as the numbers that will occur at the time of repeating the tests. The term "essentially the same" within the context means that the typical peak position and variability of intensity are taken into account (since they could normally be for any analytical technique). For example, one skilled in the art will appreciate that the peak positions will show some inter-device variability. For example, the 2-theta peak positions will normally deviate as much as 0.1 degrees. In addition, one skilled in the art will appreciate that the relative peak intensities will also show variability due to the degree of crystallinity, preferred orientation, prepared sample surface and other factors known in the art. Therefore, relative peak intensities should be taken only as a qualitative measure.

Figure 4 shows the characteristic XRPD pattern of the malonate salt. The characteristic major peaks defining this crystalline form of the malonate salt are shown in tables 8A and 8B.

Table 8APXRD peaks of Malonato saltTable 8BBasic Data ProcessingGroup nameData Name 34964BFile Name 34964: 5. F RSample Name Lot # 2587-169-17Comment Sales 11 SP 941 ># of strongest peaks 3do not. 2 Teta or 1/11 FWHM Int Integrated Intensity peak (degrees) (A) (degrees) (Count) (Count)13 17.7561 4.99119 100 0.31900 947 16049? 4.916B 5. S3423 92 0.30230 667 14513 IB.8000 • i .11634 80 0.657 0 758 23557# of Peak Data Listdo not. 2 Tithe d 1/11 FWH Intensity Int Integra of peak (degrees) (A) (degrees) (Count) (Count)1 4.7340 IB .6S124 i 0.16140 9 692 5.9215 j.4 .91 31 11 0.26430 100 17233 7.000Q 12 .61783 3 0.20Ó60 29 5634 7.5504 11 .69922 29 .0.44910 279 64895 S.1S0O 9 .65724 8 0. 2000 76 17796 10.5600 3 .37073 4 C.18660 34 6e7 11.0208 8 .02175 34 0.29110 323 44S88 11,160O 7 .92200. 30 O.56800 287 770B9 12.6400 E .9S756 16 0.25600 147 254510 1 .8400 6 .88901 20 0.27840 191 223411 1.4.5200 6 .09549 9 C .16300 8? 12B212 14. 168 c .93423 92 0.30230 867 1451313 17.7561 4 .99119 100 0.31900 947 1604914 13.3000 4. S 40S 73 0.40260 692 1265615 13.8000 4 .71634 P.O 0.65720 758 2159716 19.2077 4 .6 7 41 0.36220 387 66081 19.5800 4 .53018 63 0.34720 593 11731.18 20.0200 4. 3159 14 0..15560 130? 8319 20. 824 i .270 1 2-1 0.541 0 223 6510.70 22.0112 .0349S 6 0.477 SO 250 604021 22. 800 3 .95190 G ^ 0.45260 211 433323. 3273 3 .81023 IB 0.29470 171 27183 24.1490 3 .682 '! G: 24 0.51800 232 £ 30224 25.3900 3 .50653 44 0.43120 413 1410025. 85.30 3 .43321 44 0.279T? 419 553925 26.2380 .35378 24 0.79670 227 esio27 26.8200 3 .321 40 0. G1720 363 1367528 27.30T.7 3 .26310 14 0.49680 130 305729 27.7600 .21107 18 0 · .42.180 175 429130 28.9185 3 .0B501 27 0.5 570 258 S92431 29.3200 3 .04367 11 C .20660 104 202132 30.3600 .94173 13 0.65340 11 19433 31.1400 .86980 17 0.56000 159 425634 32.0000 2 .7S46I 3 0.14340 25 j 653 S 32.8233 .72637 10 0.29230 95 1975- 36 33.8725 .64428 7 0.5S500 64 íessFigure 5 shows the characteristic XRPD pattern of the succinate salt. The main and characteristic peaks defining this crystalline form of the succinate salt are shown in Tables 9A and 9B.

Table 9APXRD peaks of succinate saltTable 9BBasic Data ProcessingGroup nameData Name 34984AFile Name 34984A. PKRSample Name Lot # 2587-166-27Comment Sales USP < 941 ># of strongest peaks 3do not. do not. 2 Teta d I / FWHM Intensity Int Integrated peak (degrees) (A) (degrees) (Count) (Count) 10 24.9100 3.57161 100 1.13000 590 40991 18.4600 4.80243 81 0.73400 480 16639 1 .7600 4.99010 79 O .41620 464 9106# of strongest peaks 3do not. 2 Titrate l / ll FWHM Int Int. Intensity of peak (degrees) (degrees) (count) (count) 1 6.0033 .71029 7 0.64670 43 1492 2 9.4308 9.37032 12 1.24830 69 44321 11.7717 7.51169 33 1.08060 195 11923 d 14.7400 6..00500 10 0.600O0 60 42885 16.0720 5.51020 9 0.55880 56 14706 17.7600 4.99010 79 0.41620 464 91067 ia.4600 .80243 61 0, 73400 480 166398 20.4772 .33367 57 0.87020 337 138699 21. 293 4.14323 43 0.40730 252 4832 10 2 .9100 3.57161 IDO 1.13000 590 40991 11 26.1400. 0628 36 2'.92000 212 21458 12 2 .9400 3.19019 15 1.47000 66 8157 13 29.7800 2.99769 9 0.31340 53 779 14 30.9200 2.88972 7 1.28000 40 3619 1S 33.2233 2. £ 9446 8 1.08670 49 2527Figure 6 shows the characteristic XRPD pattern for the citrate salt. The main and characteristic peaks that define this crystalline form of the citrate salt are shown in tables 1 OA and 1 OB.

Table 1 OAPXRD peaks of citrate saltTable 10 BBasic process of DalosGroup nameData Name 3S614AFile Name 35614A. PKPSample Name Lotej) 27B2-76- Comment USP < 941 ># of strongest peaks 3do not. 2 Teta I / Il FWHM Integrated Inl Peak Intensity (degrees) (A) (Count) (Count)21 19.78S8 4.4B352 1.00 0.26810 1443 20S3B 30 23.8400 3.72944 55 0.33400 793 14S67 35 26.S9SS 3.34884 51 0.31810 734 11131# of Peak Data Listdo not. FWHM Integrated Int Intensity2 Teta d I / T.1(degrees) (Count) (Count) peak (degrees) (A)1 6.7400 13.10398 3 0.163GO 47 455 6.9496 12.70922 6 0.2 930 B7 B643 8.2200 10.74764 6 0.23.20 81 14144 8.4436 10.46352 18 0.22630 253 28025 9.0566 9.75662 6 0.22460 B9 12756 11.0600 7.9S341 4 0.18000 62 7627 11.2871 7.83308 16 0.23720 238 27709 11.8800 7.44345 4 0.19560 52 6039 12.0S0B 7.33833 8 0.25030 114 1167 10 13.1691 6.717S8 16 0.2217D 229 3085 1 13.9959 6.32255 25 0.23910 358 5182 12 1 .6151 6.0S6 4 33 0.26400 470 7221 13 15.4624 5.72604 25 0.30010 356 SG58 14 15, 8200 5.59740 3 0.1BB00 48 832 ib 16.9600 5.22354 26 0.27100 377 549S 16 17.3474 5.10785 46 0.26120 668 9289 17 18.0000 .92411 1 0.19920 271 3037 IB 18.2200 86514 32 0.20420 465 5224 19 18.7154 73746 4 0.21520 51 606 20 19, 2200 .61421 10 0.19200 146 2871 21 19.785 B .4B252 100 0-26810 1443 20538 22 20.0BOB .41849 38 0.193B0 546 6060 23 20.5315 32233 32 0.32250 455 7724 24 21.1200 .20320 e 0.25500 114 1667 25 21. 000 .14BE3 16 0.26760 235 305426 21.8913 4. C56B2 10 0.26260 141 2250 27 22.8001. e9713 13 0.30 ?? 181 2641 29 23,2200 3.82760 3 0.19760 50 746c.29 23.3691 3.80351 0.16040 83 625 30 23.8400 3. 2944 55 0.33400 753 14567 31 24.1800 3.67776 26 0.12300 380 4146 32 25.0296 3.55482 f, 0.32290 119 2002 33 25.6031 3.47648 7 0.2209C 102 1365 34 26.2800 3.38845 26 0.30180 379 5681 35 26.5965 3.348B4 5 ) 0.31B10 734 1113136 27.4B4B 3.24255 6 0.20170 1222 37 28.1600 3.1GC36 4 0.05340 441 8 28.5989 3.11875 46 0.33280 666 12909 39 29.0484 3.07151 or 0.23960 127 1440 40 25.5600 2.0195C 20 0.48340 290 6624 41 29.8RO0 2.98789 16 0.14600 2012 42 30. 400 2.93413 0, 09340 274 43 30.7800 2.90254 11 C.10020 737 44 31.0600 2.87701 6 0.19060 350445 31.3400 2. E5195 0.18000 B02 46 31.5800 2.83082 0.12960 357 47 31. B602 12 i l 2604 46 32.2200 2.? 503 4 0.08.580 55 4B2 no. 2 Teta d I / Il FWHM Intensity Int Integrated peak (degrees) (A) (degrees) (Count) (Count) 49 33.08O0 2.70580 3 0.09060 5 386 50 33.2668 2.69103 5 0.12920 65 453 51 33. S21 2.66571 7 0.36940 108 2006 52 34.3755 2.60673 6 0.15550 B3 1053All mentions of the literature and prior patents are expressly incorporated in this manner as a reference in the places where they are mentioned. The sections or pages mentioned specifically about the works mentioned above, are incorporated with specificity as a reference. Although the present invention has been described in sufficient detail to enable one skilled in the art to make and use the subject or matter of the claims set forth below, it may also be contemplated that certain modifications of the appended claims may be made and remain still within the scope and spirit of the present invention.

Claims (32)

1. A citrate salt of formula I or a hydrate thereof: (l)

2. The salt or hydrate as described in claim 1, characterized in that it is crystalline.

3. The salt as described in claim 2, characterized in that the spectral-spacing absorption bands d are obtained from an X-ray powder diffraction pattern thereof or essentially the same 4.48, 3.12 and 6.05 angstroms.

4. The salt as described in claim 2, characterized in that the absorption bands of the diffraction angle 2-teta are obtained from the X-ray powder diffraction pattern thereof or essentially the same 19.81, 28.63 and 14.64 degrees.

5. The hydrate as described in claim 2, characterized in that it is partially or completely hydrated.

6. The salt as described in claim 2, characterized in that it is anhydrous or essentially anhydrous.

7. A pharmaceutical composition comprising a therapeutically effective amount of the salt or hydrate as described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition as described in claim 7, characterized in that it also comprises an additional therapeutic agent.

9. The pharmaceutical composition as described in claim 8, characterized in that the therapeutic agent is an anti-HIV drug.

10. The pharmaceutical composition as described in claim 7, characterized in that the pharmaceutical composition is a unit dosage form.

11. The pharmaceutical composition as described in claim 10, characterized in that the unit dosage form is a tablet.

12. A pharmaceutical composition prepared by combining the therapeutically effective amount of the salt or hydrate as described in any one of claims 1 to 6, with a pharmaceutically acceptable carrier or excipient.

13. The use of the salt or hydrate as described in any one of claims 1 to 6 to prophylactically treat or prevent an HIV infection.

14. The use of the salt or hydrate as described in any one of claims 1 to 6 for the manufacture of a medicament for prophylactically treating or preventing an HIV infection.

15. The use as described in any of claims 13 to 14, characterized in that the salt or hydrate are present in the form of a pharmaceutical composition as described in any of claims 7 to 12.

16. A succinate salt as described in formula II or a hydrate thereof: (ID

17. The salt or hydrate as described in claim 16, characterized in that it is crystalline.

18. The salt as described in claim 2, characterized in that the spectral spacing-d absorption bands are obtained from an X-ray powder diffraction pattern of the same or essentially the same 3.57, 4.80 and 4.99 angstroms.

19. The salt as described in claim 2, characterized in that the absorption bands of the diffraction angle 2-teta are obtained from the X-ray powder diffraction pattern thereof or essentially the same 24.91, 18.46 and 17.76 degrees.

20. A pharmaceutical composition comprising a therapeutically effective amount of the salt or hydrate as described in any of claims 16 to 19 and a pharmaceutically acceptable carrier or excipient.

21. The pharmaceutical composition as described in claim 20, characterized in that it comprises an additional active ingredient.

22. A pharmaceutical composition prepared by combining the therapeutically effective amount of the salt or hydrate as described in any of claims 16 to 19 with a pharmaceutically acceptable carrier or excipient.

23. The use of the salt or hydrate as described in any of claims 16 to 19, to prophylactically treat or prevent an HIV infection.

24. A malonate salt of formula III or a hydrate thereof: (III)

25. The salt or hydrate as described in claim 24, characterized in that it is crystalline.

26. The salt as described in claim 25, characterized in that the spectral spacing-d absorption bands are obtained from an X-ray powder diffraction pattern thereof or essentially the same 4.99, 5.93 and 4.72 angstroms.

27. The salt as described in claim 25, characterized in that the absorption bands of the diffraction angle 2-teta are obtained from the powder diffraction pattern of X-rays thereof or essentially the same 17.76, 14.92 and 18.80 degrees.

28. A pharmaceutical composition comprising a therapeutically effective amount of the salt or hydrate as described in any of claims 24 to 27 and a pharmaceutically acceptable carrier or excipient.

29. The pharmaceutical composition as described in claim 28, characterized in that it comprises an additional active ingredient.

30. A pharmaceutical composition prepared by combining the therapeutically effective amount of the salt or hydrate as described in any of claims 24 to 27 with a pharmaceutically acceptable carrier or excipient.

31. The use of the salt or hydrate as described in any of claims 24 to 27, to prophylactically treat or prevent an HIV infection.

32. A composition or method as described in the present invention.